This invention relates to synthetic peptides having hypocalcemic action similar to that of natural calcitonins of various species. It relates specifically to synthetic calcitonin peptides having a hypocalcemic potency greater than that of human calcitonin and/or having less immunogenicity in humans than calcitonins from foreign species. The invention also relates to synthetic analogues of calcitonin as well as other naturally occurring bioactive peptides having a cyclic peptide structure, wherein the synthetic cyclic strucutre has improved resistance to metabolic attack. It relates as well to processes for producing these synthetic peptides.
Calcitonins are 32-amino acid peptide hormones invovled in the regulation of calcium metabilism. Calcitonin participates with parathyroid hormone in the regulation of bone metabolism and the homeostatic regulation of blood calcium levels according to mechanisms that are not completely understood. Normal bone metabolism comprises a balance of osteolytic resorption and osteoblastic formation of new bone to fill the resorption spaces. Calcitonin appears to oppose the osteolytic activity of parathyroid hormone, acting directly to inhibit bone resorption by altering osteoclastic and osteocytic activity. Calcitonin may also enhance new bone formation by stimulation of osteoblasts.
Bone resorption causes a release of clacium and alkaline phosphatase into the circulation, and the appearance of urinary hydroxyproline, resulting from the breakdown of collagen-contining bone matrix. According to physiological mechanisms, elevated serum calcium levels promote the secretion of calcitonin, which has a hypocalcemic effect. In normal individuals, bone resorption is minimal, and exogenous calcitonin has no hypocalcemic effect.
Many diseases in man, including not only those associated with bone resorption, but those related to other disorders, including malignancy, are marked by hypercalcemia, the persistence of which can be life-threatening. Exogenous calcitonin has proved to be a valuable therapeutic agent in treating these disorders. Calcitonin therapy is thus effective in diminishing hypercalcemia in patients with hyperparathyroidism, idiopathic hypercalcemia of infancy, Vitamin D intoxication, and osteolytic bone metastases. It similarly diminishes the hypercalcemia that accompanies malignancies with or without metastasis, and that of multiple myeloma.
Calcitonin is also effective in treating disorders wherein bone turnover or resorption is accelerated, but changes in serum calcium levels are not detected. One important disease of this type is osteoporosis, particularly postmenopausal type, wherein there is a progressive loss of bone mass. The efficacy of calcitonin in osteoporosis is determined by its ability to increase total body calcium. Paget's disease (osteitis deformans) is a disorder characterized by excessive resorption of bone accompanied by the imbalanced formation of new (pagetic) bone which lacks the characteristic architecture of normal bone. Calcitonin reduces the elevated serum levels of alkaline phosphatase and urinary hydroxyproline seen in individuals with this disease. Benefits of calcitonin therapy in Paget's disease are indicated by radiologic evidence of bone remodeling, correlated with a reduced number of osteoclasts seen in bone biopsies, consistent with a decrease in bone resorption. Calcitonin also provides relief from the pain and tenderness associated with the disease.
Calcitonins are found in a variety of vertebrate species including mammals, birds and fish. The hormone is secreted by the C cells, which are localized in the thyroid gland of mammals, and in the ultimobranchial glands in the lower vertebrates.
Human calcitonin (hCT) has the following amino acid sequence: ##STR1##
Calcitonin shows considerable divergence in amino acid sequence between lower vertebrates and higher vertebrates. However, highly conserved residues are clustered at the two ends of the calcitonin molecule believed to be important for biological activity. For example, a 1-7 disulfide bridge and a C-terminal proline amide are invariate among all species. Several other invariate amino acid residues occur at the N-and C-terminal ends. The middle portion of the molecule, positions 10 to 27, which is thought to control the potency and duration of the peptide, is quite variable in amino acid composition. Breimer, L. H., MacIntyre, I., and Zaidi, M., Biochem. J. 255:377.varies.390 (1988) have reviewed the structures and biological properties of calcitonin peptides from various species and this information is hereby incorporated by reference.
Calcitonins of certain non-human species appear to be more potent in humans than human calcitonin. Calcitonins that are ultimobranchial in origin, such as salmon, eel, and avian are more potent than thyroidal calcitonins, such as human or porcine hormones. Salmon, eel, porcine and human calcitonins are currently in clinical use for the treatment of Paget's disease, osteoporosis and the hypercalcemia of malignancy.
The correlation of potency with the structure of the calcitonin peptides is not well understood. Improved potency may be due to an amino acid sequence which permits a peptide conformation that is more favorably bound to the hormone receptor (Marx et al., Science 178:998-1001 (1972). A conformation that is more flexible, a feature provided by smaller less bulky amino acids, has been determined to affect biological activity (Epand et al., Biochemistry 25:1964-1968(1988)). The identical biological potencies of eel and salmon calcitonin may accordingly be explained on the basis of similar primary structures and similar flexibility.
An alternative basis for the relatively greater potency of non-human calcitonins may be that the amino acid sequences of these calcitonins, characteristic of particular species, offers greater resistance to metabolic degradation in the human body than human calcitonin, and for this reason has a more persistent effect (Habener et al., Nature(London) 232:91-92 (1971)). For example, salmon calcitonin remains potent for about six hours after administration, while human calcitonin remains potent for about two hours.
In spite of their higher potency, however, the calcitonins from other species, such as the ultimobranchial calcitonins, are not entirely satisfactory for human clinical use, primarily because the variable, poorly conserved middle portion of non-human calcitonins acts as an immunogen in vivo. The resulting antibody production can therefore limit their usefulness.
After administration to man by subcutaneous injection, all the natural calcitonins have a relatively short half life because, in spite of species differences which act to retard proteolysis by plasma enzymes, they are subject to rapid renal and tissue clearance as well. Also, because the activity of natural calcitonins depends on an intact disulfide bond between the cysteine groups at positions 1 and 7, the reduction of this unstable bond in vivo rapidly converts biologically active peptides to an inactive form.
It would be useful to have calcitonin peptides which are more effective in clinical use either because of greater stability in vivo, and/or higher potency and longer duration of action than the native hormones. It would also be useful to have calcitonin peptides which are less immunogenic than the native hormones.
It would also be useful to have calcitonin peptides which are convenient and inexpensive to synthesize and that can be stored for a period of time prior to clinical use.
Furthermore, analogues with increased lipophilicity and hydrophobicity could have altered pharmacokinetics and possess improved parenteral, nasal or oral bioavailability. It would, further, be advantageous to extend these advantages to other bioactive peptides having structural similarities to calcitonin.
Accordingly, it is an object of the invention to provide synthetic calcitonin peptides which are safer and more effective in the treatment of human disease than native calcitonins from human and other species.
It is also an object of the invention to provide synthetic analogues of calcitonin peptides and other bioactive cyclic peptides having i-ncreased chemical and enzymatic stability.
It is also an object of the invention to provide synthetic cyclic peptides which are inexpensive to manufacture and which are stable under conventional storage conditions.
It is also an object of the invention to provide methods for the synthesis of these cyclic peptides.